Quantification of Microbial Fluorescent Sensors During Live Intracellular Infections

The interaction of pathogens with their eukaryotic hosts during intracellular growth is critical to many diseases. However, the relative scarcity of pathogen biomolecules versus the abundant host biomolecule concentration can make quantitative evaluation of pathogen intracellular responses difficult. Recent years have seen an explosion in utilization of fluorescent proteins to serve as transcriptional reporters and biosensors for quantification of pathogen responses. Here, we describe a method to establish a fluorescent assay quantifying pathogen behavior during intracellular infection and to quantify these results at a single cell level. The sensitivity of these fluorescent assays permits the live observation of changing pathogen responses, while the ability to measure at a single cell level uncovers subpopulations of pathogens whose existence may be missed during the population-level assays often required to accumulate sufficient pathogen biomolecules for analysis.